The present disclosure relates generally to systems and methods for functional imaging, and, in particular, to systems and methods for fluorescence imaging in turbid media.
Imaging based on fluorescence provides a powerful tool for non-invasive diagnosis of disease in living animals or human subjects, as well as facilitates investigations into many biological processes In fluorescence, incident excitation photons of appropriate wavelengths are absorbed by a sample including fluorescent molecules, or fluorophores, which then emits light at wavelengths that depend upon the energy level arrangement of each fluorophore. On account of a high specificity, fluorescence imaging permits functional contrast resulting from both intrinsic auto-fluorescence and extrinsic fluorescence, using both absorption and emission characteristics unique to particular molecular structures. When appropriately generated and analyzed, optical signals can provide valuable information with respect to the fluorophores embedded in a turbid medium, such as environment, location, or distribution, as well as a contrast between species with different emission spectra. As such, development of disease-specific fluorescent markers and genomic reporters has prompted many concurrent advances in optical imaging techniques using fluorescence.
Fluorescence imaging techniques are, in general, confounded by several factors. First, typical fluorophores include a small Stokes shift, which describes the separation between absorption and emission spectra peaks. Usually optimal detection of fluorescence necessitates choosing filter wavelengths very close to those of the excitation. Coupled with the poor rejection ratio of typical emission filters, this implies a significant leakage of the excitation signal into the fluorescence signal. In the case that fluorophore signal is very intense, this is may not be a major concern. However, since a majority of fluorophores have a low emission quantum yield, which describes the conversion efficiency from excitation to fluorescence, fluorescence signals can be easily overwhelmed by the excitation signal, if the latter is not filtered out efficiently.
A second confounding factor includes the measurement of intrinsic fluorescence lifetimes of fluorophores embedded in scattering tissue. Optical signal detection techniques typically involve three main approaches, namely time-domain (TD) detection using pulsed sources (typically in the femto-second to pico-second pulse duration range), frequency-domain (FD) detection using modulated sources (typically in the MHz frequency range) and continuous wave (CW) detection using steady state light sources. Of these, the TD approach is most comprehensive since short pulsed sources contain all modulation frequencies, including the zero-frequency component, and facilitate direct lifetime estimation by fitting a decay portion of a spatio-temporal optical signal. By contrast, FD techniques require reconstruction algorithms to recover the true lifetime.
However, even for measurement approaches using TD, unless the temporal decay time constant (lifetime) of fluorescence is longer than about one nano-second (equivalent to intrinsic diffusion timescales), decay times are affected by the optical properties, such as intrinsic absorption and scattering, of the surrounding turbid medium. Therefore, recovery of fluorophore intrinsic lifetimes becomes ambiguous and necessitates complicated reconstruction algorithms that include inversion of coupled differential equations describing fluorescence propagation in tissue, and may require prior knowledge of tissue absorption and scattering. Such approaches can be ill-posed, and may provide additional complexities.
Therefore, there is a need for systems and methods for detecting fluorescence lifetimes and decay profiles in turbid media.